This proposal describes experiments to identify proteins involved in eucaryotic nucleic acid metabolism, to purify them, and to elucidate their function. Initial experiments are aimed at identifying the role of DNA- dependent ATPases in nucleic acid metabolism. Monoclonal antibodies that recognize at least two distinct forms of a calf thymus, DNA-dependent ATPase will be used to define the integrity of that enzyme. Both forms of the enzyme recognize primer-template junctions (a DNA polymerase substrate) as effectors for ATP hydrolysis. Consequently, this enzyme is expected to play a role in DNA replication. The proposed experiments are aimed at: 1) using the monoclonal antibodies to develop a method of purification for the intact DNA-dependent ATPase. 2) Defining the relationship of this ATPase to other chromatographically distinct DNA-dependent ATPases. 3) Defining the role of this enzyme in cellular processes through the use of monoclonal antibodies in permeabilized cell systems and using cell cycle synchronization. 4) Identifying additional proteins with which the enzyme(s) interacts. While there has been considerable difficulty in the study of eucaryotic DNA binding proteins and their role in nucleic acid metabolism in the past, this project will use the latest technologies in order to accomplish its goals. These technologies include production and use of monoclonal antibodies, immunoaffinity and specialized affinity chromatography, and an ultraviolet laser cross-linking technique which effectively """"""""freezes"""""""" protein-nucleic acid interactions with a five nanosecond pulse of light. An understanding of specific protein-nucleic acid interactions will aid us in better understanding processes such as DNA replication and may give us an understanding of where normal systems go awry resulting in various genetic disorders, including inability to conceive, birth defects, and various cancers. The information gleaned from identification of specific protein-nucleic acid interactions may be critical for the design of drugs that prevent or control certain disease states.
